In the last decade, there has been growing appreciation of adipose tissue (AT) inflammation in obesity, and its link to insulin sensitivity. Te prevalence of insulin resistance in our population makes it imperative that we come to a better understanding of the role of immune cells in maintaining healthy AT. Macrophages are the predominant immune cell in AT and are known to change to an inflammatory phenotype in response to unhealthy lipid accumulation in obesity. Lean AT contains a resident population of anti-inflammatory M2-like AT macrophages (M2 ATMs), whose role in the maintenance of a healthy lean state is not well understood. Our lab has recently identified a MFehi sub-population of M2 ATMs, with a two-fold increase in intracellular iron content and increased expression of genes involved in iron uptake (e.g. CD163), storage and release. This population composes 25% of M2 ATMs in lean AT and also exists in obese mouse and human AT. Previous studies have demonstrated that adipocytes require iron for adipogenesis, but excess iron is cytotoxic and leads to systemic changes in insulin sensitivity. Therefore, healthy adipocytes must maintain a narrow range of intracellular iron concentrations. It remains unknown whether MFehi ATMs contribute to adipocyte iron homeostasis. We hypothesize that MFehi ATMs modulate iron availability to adipocytes, and thereby impact insulin sensitivity of lean and obese AT. To identify possible iron exchange between MFehi ATMs and adipocytes, we will co-culture these cells after iron-loading treatment and from genetic models of iron overload. In initial studies, we have successfully quantified increased intracellular iron in macrophages after treatment with ferrous ammonium citrate and the chelator 8-hydroxychloroquine. We also propose to deplete MFehi ATMs in vivo in mice using CD163-targeted clodronate-containing liposomes. Depletion will allow us to determine the impact of MFehi ATMs on the systemic metabolic state. Our preliminary studies with fluorescent calcein-loaded liposomes in the peritoneum have revealed CD163-targeted liposomes as a novel technique to specifically target ATMs. Lastly, comparative metabolomics analysis of MFehi and MFelo ATMs will serve as an unbiased hypothesis-generating approach to characterizing the MFehi phenotype. From our current understanding of iron in cell function, we expect to find changes in mitochondrial and/or lipid oxidation pathways in the MFehi population. The studies in this proposal will allow us to define the role of the MFehi population in maintaining healthy adipocyte iron concentrations and, thereby, insulin sensitivity.